Covalent modificaition of juvenile hormone binding proteins by photoaffinity labeling: An unexpected gel shift effect

Abstract

The 32 kD juvenile hormone binding protein (JHBP) and two 80 kD proteins in larval Manduca sexta hemolymph were labeled with [3H]FDK, a photoaffinity analog of methyl farnesoate (MF). The labeling could be completely displaced by a 30-fold excess of either MF or JH II, demonstrating that [3H]FDK binds specifically to the JH binding sites of the 32 kD JHBP and the 80 kD proteins. In addition, a high molecular-mass protein was labeled with [3H]FDK; labeling could be displaced by excess MF but not by JH II, demonstrating the selectivity in binding MF. The 32 kD JHBP also appeared to weakly bind the potent juvenoid, methoprene, at the JH binding site. Covalent modification by [3H]FDK induced a change in the apparent size and the isoelectric point of the JHBP. These changes were not induced by substrate alone, nor by UV irradiation alone. The same effect was also observed during labeling with [3H]MDK, an analog of methoprene. These data provide an important caveat for anticipating artifactual changes of protein properties during chemical or photochemical affinity labeling experiments. The molecular dimensions of [3H]FDK more closely resemble those of JH II than those of [3H]EHDA, a photoactivatable analog of JH II. We suggest that covalent modification by a diazoketone photolabel involves a hydrophilic amino acid important in the recognition of the ester group of JH. © 1994 Wiley-Liss, Inc.